Method to prevent fertilization in mammals by administering a single dose of zona pellucida derived antigens, liposome and adjuvant

ABSTRACT

A vaccine for the immunocontraception of mammals is described. The vaccine consists of zona pellucida antigens and an adjuvant encapsulated in a liposome delivery system. The liposome delivery system allows for the slow release of antigen resulting in a prolonged immune response. In particular, after a single injection of the vaccine, levels of anti-zona pellucida antibodies were detected for up to 22 months in seals. Thus, the vaccine according to the present invention is effective after a single dose and is therefore very useful in immunocontraceptive protocols.

This application is a continuation of application Ser. No. number08/347,348, filed Dec. 5, 1994, now abandoned, which is the nationalphase of PCT/CA93/00239, filed Jun. 7, 1995 1993, which is acontinuation-in-part of U.S. patent application No. 07/892,807, whichwas filed on Jun. 5, 1992 and is now abandoned.

FIELD OF THE INVENTION

The present invention relates to a vaccine composition for theimmunocontraception of mammals.

BACKGROUND OF THE INVENTION

There is a real need for population control in several species ofdomestic and wild animals. Methods such as surgical sterilization, ormore drastically, culling are generally not acceptable or even allowablein most countries. For example, in Canada the culling or harvesting ofseals was prohibited in the early 1980's, resulting in an increased sealpopulation from 10,000 in 1978 to approximately 45,000 at present.Increases in harp seal populations have been much greater.Unfortunately, the increasing population of seals is eating away at thediminishing fish stocks which poses serious problems for the fishingindustry. The seals also contain parasites such as seal worms that theypass on to the fish. In 1986 it was estimated that the cost to thefishing industry of removing seal worms from fish by hand was upwards of$30 million a year. Therefore, it is highly desirable to develop aneffective form of contraception in mammals, such as seals, in order toeffectively control the population growth of certain mammals.

One form of contraception for mammals has involved immunocontraceptionusing glycoproteins isolated from the zona pellucida, a covering whichsurrounds oocytes. The zona pellucida glycoproteins (hereafter referredto as ZP) provide an attachment site for sperm. Immunocontraception withZP results in the production of antibodies to ZP which cause (a) analteration of the nature of the ZP membrane of ova, thereby inhibitingsperm entry, (b) an inhibition of implantation of fertilized ova intothe uterus and (c) decreased ovarian follicular differentiation(Henderson, C. J., M. J. Hulme and R. J. Aitken. 1988. Contraceptivepotential of antibodies to the zona pellucida. J. Reprod. Fert. 83:325-343). Immunocontraception has been induced with both zona pellucidaglycoproteins and epitopes of these glycoproteins which have beensequenced, synthesized and coupled to carrier proteins (Millar, S. E.,S. M . Chamow, A. W. Baur, C. Oliver, F. Robey and J. Dean. 1989.Vaccination with a synthetic zona pellucida peptide produces long-termcontraception in female mice. Science 246: 935-938). The use of ZPglycoproteins for immunocontraception has several advantages over othercontraception methods. Firstly, ZP glycoproteins are unique to thefemale reproductive system and therefore, anti-ZP antibodies likely havelittle or no effect on other tissues. Secondly, the infertility causedby anti-ZP antibodies is reversible, although, hyper- immunization maycause permanent sterility.

Previous studies have shown that in order to effect immunocontraception,multiple injections of ZP were necessary (Kirkpatrick, J. F., I. K. M.Liu and J. W. Turner. 1990. Remotely-delivered immunocontraception inferal horses. Wildl. Soc. Bull. 18: 326-330). Multiple injections areclearly not practical for wild populations as it entails recapturing thesame wild animal each time an injection is necessary. Multipleinjections are also cumbersome in any situation.

Therefore, it is desirable to develop an immunocontraceptive vaccinewhich would be effective for long periods following a single injectionin an efficient delivery system.

Liposomes have been used to carry drugs to sites of inflammation andinfection or in some cases tumours. Liposomes are microscopic spherescomposed of either a single or multiple concentric bilayer sheets, andrange in size from a nanometer to several micrometers in diameter. Thesebilayer sheets can be formed from a wide variety of phospholipids invaried formulations. Cholesterol can be included in the bilayer in orderto increase the bilayer strength and reduce the leakage of materialsencapsulated within the entrapped aqueous interior. A vast array ofcompounds can be associated with liposomes, including small molecules,drugs, proteins, and nucleic acids. Liposome-associated compounds can beencapsulated within the aqueous interior of the liposome (i.e. betweenthe bilayer sheets), integrated into the bilayer, or adsorbed orattached to the bilayer surface. The location depends upon theproperties of the associating compounds as well as the procedures usedfor the formation of the liposome.

A liposome based vaccine system has been described for immunizationagainst human malaria (Fries et al, 1992. Liposomal Malaria Vaccine inhumans: A safe and potent adjuvant strategy. Proc. Natl. Acad. Sci. USA.89: 358-362). In this system, a recombinant malaria protein derived fromPlasmodium falcioarum was encapsulated into liposomes and injected intomale volunteers. However the results indicate that at least threeinjections of the vaccine were required in order to produce an elevatedantibody response.

SUMMARY OF THE INVENTION

The present invention relates to an immunocontraceptive vaccinepreparation which comprises zona pellucida antigens incorporated into aliposome delivery system. The liposome system effects the slow releaseof antigen resulting in an extended period of antibody production andthereby an extended period of contraception. Therefore, the presentinvention provides a method to achieve immunocontraception of mammalsusing a single injection of zona pellucida glycoproteins.

Accordingly, the invention provides a vaccine composition for theimmunocontraception of a mammal which comprises a zona pellucida derivedantigen incorporated into a liposome system.

The invention further provides a vaccine composition capable of inducingthe production of antibodies to a zona pellucida antigen, saidcomposition comprising a zona pellucida derived antigen incorporatedinto a liposome system.

In one embodiment of the present invention, the liposome vaccinecomposition is freeze-dried and incorporated into a BALLISTIVETbiobullet. Such an embodiment makes the vaccine easier to deliver to theanimal.

In another aspect, the invention provides a method of preventingfertilization in a mammal which comprises parenterally administering aneffective amount of the above-described vaccine.

DETAILED DESCRIPTION OF THE INVENTION

Isolation and purification of zona pellucide glycoproteins

Zona pellucidaglycoproteins were isolated and purified as described byYurewicz (in Yurewicz, E. C., A. G. Sacco, and M. G. Subramanian,Structural Characterization of the Mr=55,000 Antigen (ZP3) of PorcineOocyte Zona Pellucida, The Journal of Biological Chemistry 262: 564-571(1987). Porcine ovaries were homogenized and the oocytes recovered fromthe homogenate by sieving through nylon screens of decreasing pore size(500, 350, 200, 175, 100 and 40 μm). The oocytes were homogenized with aglass-teflon apparatus and the homogenate was passed through a 40 umscreen to collect the fractured zonae. The fractured zonae were washedwith buffer and recollected on the 40 um screen. Zona pellucidaglycoproteins were solubilized by incubating the zonae in a water bath(73° C. for 20 minutes). The fraction obtained (solubilized intact zonapellucida glycoproteins, SIZP) was shown to be at least 95% pure bycomparison to a reference standard of ZP using an ELISA assay.

ZP3 was purified from SIZP as described by Yurewicz et al (1987). ZP3 isone of three glycoproteins that make up the mealis zona pellucida. ZP3is the major macromolecular component of the oocyte zona pellucida andhas been shown to be the receptor for sperm.

Porcine ZP has been used to effect immunocontraception in a variety ofmammals. Porcine ZP pellucida was used in the present studies forseveral reasons. Firstly, a comparison of the reactivity of ZP from fivemammalian species to rabbit antiserum against porcine ZP indicated thatpig ZP was recognized best followed by dog ZP while rat and cat ZPreacted poorly (Maresh, G. A. and B. S. Dunbar. 1987. Antigeniccomparison of five species of mammalian zonae pellucida. J. ExperimentalZoology 244: 299-307). Since seal and dog are closely related, (Berta,A., C. E. Rae, A. R. Wyss, 1989, “Skeleton of the oldest known pinnipedEnaliarctos mealsi”, Science 244: 60-62), anti-porcine ZP antibodieswill bind to seal zonae. Secondly porcine ZP can also be easily obtainedfrom slaughterhouse pigs. Thirdly, pigs ovulate multiple oocytes andthus provide a rich source of zona pellucida.

EXPERIMENT 1

Production of Anti-ZP Antibodies in Rabbits

Protocol.

Rabbits were injected i.m. (two rabbits for each treatment, details ofeach treatment are given in Table 1) as follows (a) ZP in threeinjections at monthly intervals, (b) ZP with Freund's complete adjuvantplus two boosters with ZP in Freund's incomplete adjuvant at 4 and 8weeks and (c) ZP with muramyl dipeptide adjuvant (Sigma chemicals). In asecond series, rabbits were immunized as above but ZP, plus adjuvantwhere applicable, were encapsulated in liposomes and in all cases in thesecond series only a single injection was administered. Preimmunizationserum samples were taken from all rabbits. Titers of antibodies weremeasured by Elisa assay using both SIZP and ZP3 as antigen.

Results

The results of this study are illustrated in Table 1. Immunization ofrabbits with ZP (no adjuvant) raised a low antibody titer after thethird injection. A single injection of ZP in liposomes with no adjuvantraised no detectable antibodies. Use of muramyl dipeptide adjuvant didnot stimulate antibody production regardless of being encapsulated inliposomes. Use of Freund's adjuvant stimulated antibody production tohigh levels after 69 days when the antigen was administered by 3injections 4 weeks apart or as a single injection in liposomes. However,titers were higher using the former procedure. Measurement of antibodiesdirected specifically against ZP 3 indicated that antibody titers weresimilar after three injections without the use of liposomes as they werewith a single injection with liposomes.

Therefore, these results indicate that a vaccine preparation containingZP in combination with Freund's adjuvant and encapsulated into aliposome system can induce significant anti-ZP antibody levels forextended periods of time after a single injection.

TABLE 1 Effect of Encapsulating ZP in Liposomes on Anti-ZP AntibodyProduction in Rabbits Time Anti-ZP titer* (days) Ag a b c d e f PRE SIZP0 0  0 0 0 0 0 0    0  0 0   0   27 SIZP 2 0 12  2 0 0 0 0.5 22 36 0  0.7 55 SIZP 12  3 96 68 2 1 0 0   50 50 0.3 0.6 69 SIZP 24  24  184 130  — — — — 84 85 — — 69 ZP3 16  15  97 46 — — — — 74 64 — — *A valueof 100% indicates the serum being tested has the same titer as a pooledstandard serum from rabbits rendered infertile by immunization withSIZP. a - each rabbit was injected with SIZP (20 μg) in 0.5 ml saline.b - each rabbit was injected first with SIZP (20 μg in a mixture ofsaline (0.25 ml) and Freund's complete adjuvant (0.25 ml) followed bytwo boosters of SIZP (20 μg) in a mixture of saline (0.25 ml) andFreund's incomplete adjuvant (0.25 ml). c - each rabbit was injectedwith SIZP (20 μg) with muramyl dipeptide (5 μg) in saline (0.5 ml). d -each rabbit was injected with SIZP (20 μg) encapsulated in soya lecithinliposomes containing phospholipon 90G (Nattermann Phospholipid Co.,Cologne, Germany, 0.1 g), cholesterol (0.01 g) and saline (0.5 ml).Liposomes were prepared according to U.S. Pat. No. 4,485,054, which isincorporated herein by reference. Liposomal products containing ZP andadjuvants however can be prepared by many other methods presently knownand used for manufacturing liposomes. e - each rabbit was injected withSIZP (20 μg) encapsulated in liposomes as for d (0.25 ml) in Freund'scomplete adjuvant (0.25 ml). f - each rabbit was injected with SIZP (20μg) encapsulated in liposomes as for d (0.5 ml) containing muramyldipeptide (5 μg). PRE = preimmunization.

EXPERIMENT 2

Immunization of Captive Seals

Protocol.

Captive female grey seals (13) were divided into six groups of twoanimals each, except for group d which comprised three animals. Thegroups were immunized with a single injection i.m. as follows:

(a) ZP (15 μg) in liposomes with Freund's complete adjuvant (FCA)

(b) ZP (90 μg) in liposomes with FCA

(c) ZP (90 μg) in FCA

(d) ZP (90 μg) in liposomes with TITERMAX*

(e) ZP (90 μg) in TITERMAX

(f) ZP (90 μg) in liposomes with BCG adjuvant in water

All animals received in injection having a volume of 1.0 ml. BacillusCalmette-Guerin or BCG is one component of FCA. Liposomes wereformulated as previously described. TITERMAX (0.5ml) mixed with saline(0.5 ml) containing SIZP or liposomes (0.5 ml) containing SIZP asdescribed by the manufacturer. TITERMAX (Cytrx Corporation, Norcross,Ga.) is a new adjuvant system which claims to induce higher titers ofantibodies than FCA with a single injection while being less toxic toanimals.

Results

The results of the immunization of the seals are shown in Table 2. The1st and 2nd line of each group refers to results for two seals receivingthe same treatment, that is, each formulation was tested in two seals,except for the ZP/TITERMAX/liposomes formulation which was tested inthree seals. As can be seen from this table, the anti-ZP titer wasgreatest for groups (a) and (b) wherein zona pellucida was incorporatedinto a liposome system. Antibody titers were detected up until 22 monthsfrom immunization in the three seals from groups (a) and (b). (Thefourth seal was returned to the wild at 11 months.) Without liposomes,group (c), antibody titers dropped off after 5 months. Substituting BCGfor Freund's complete adjuvant was not effective in eliciting hightiters (group f). The new adjuvant system, TITERMAX, was also noteffective (groups (d) and (e)).

Therefore, the results of this study demonstrate that incorporation ofthe ZP and adjuvant into a liposome system results in a prolongedanti-ZP response after a single injection.

EXPERIMENT 3

Immunization of Harbour Seals

Twelve harbour seals, divided into three groups of four, were immunizedwith ZP in a single injection (Table 3). The seals were immunizedimmediately after giving birth. Samples were taken in the two or threeweek period until the seals left the pupping area. The results are shownin Table 3. Immunization (with 30 and 90 μg ZP) of harbour seals thathad just given birth resulted in anti-ZP titers of only 9% (range 6 to12%) of immunocontraceptive levels after 3 weeks. As discussedpreviously, anti-ZP antibodies can act

TABLE 2 Anti-ZP Antibody Production by Captive Seals. Anti-ZP titer (%of standard serum)* Time (months) Antigen 1 2 3 4 5 6 7 8 9 10 11 12 1316 18 20 22 (a) ZP (15 μg) 4 70 60 155 146 141 136  94  34  20 — — — — —— — FCA/liposomes 3 92 166  216 192 182 224 185 150 117 135 95 87 23 3520 18 (b) ZP (90 μg) 1 66 206  186  74 169 125 103  64  67  59 68 20 10 3  5  5 FCA/liposomes 18  78 230  227 230 247 223 164 127 122 115 74 3114 10  7  4 (c) ZP (90 μg) 51  253  201  145  53 FCA 8 200  171  110  35(d) ZP (90 μg) 3 16  9  2  1 TITERMAX/ — 22  8  2  3 liposomes 25  28 20 7  2 (e) ZP (90 μg) 10   9 25  30  14  32 — — — — — TITERMAX 3 36 22 59  7  20 — — — — — (f) ZP (90 μg) 0.1 0.3 0.1 0.1  0 — — — — — —BCG/liposomes 0.4 0.2 0.1  0 0.1 — — — — — — ZP in this table indicatesSIZP *A value of 100% indicates the serum being tested has the sametiter as a pooled standard serum from rabbits rendered infertile byimmunization with SIZP. — = no samples, seals returned to wild.

as an immunocontraceptive by inhibiting sperm penetration of ova or byinhibiting implantation of fertilized ova into the uterus. Since sealsbreed about 3 weeks after giving birth, it is possible that the abovelevels of anti-ZP antibodies would not affect sperm penetration of theoocytes. Immunization directly after giving birth may, however, havesome effect on the implantation of the embryo since seals have a delayedimplantation of up to three months.

TABLE 3 Production of anti-ZP antibodies by harbour seals immunized withZP in a single injection at three doses Anti-ZP titer (expressed as a %of a standard serum)* ZP Time (weeks) (μg) Animal 2 3 15 F — 5.9 F 1.20.4 F 0.7 2.7 F 4.8 — 30 F 0.9 — F 1.8 6.5 F — 12.0  F 18.0  — 90 F 1.16.9 F 0.4 — F 4.7 — F 1.6 11.0  F = adult female — = no sample *A valueof 100% indicates the serum being tested has the same titer as a pooledstandard serum from rabbits rendered infertile by immunization withSIZP.

EXPERIMENT 4

Immunization of Grey Seals

Field studies of grey seals on Sable island have been conducted and arestill in progress. Grey seals go to Sable island every year to have pupsand breed and were therefore used in order to study theimmunocontraceptive ability of the vaccine preparation. One group offemale grey seals (approximately 100 animals) was immunized during theJan. 1992 breeding season with liposomes in FCA (control) while theother group of 100 animals was immunized with ZP in liposomes in FCA. InJan. 1993 the seals that returned to Sable Island were tested foranti-ZP titers. The results of this study are shown in Table 4. Asexpected, the control group of seals had antibody titers of zero. Theexperimental group, on the other hand, had appreciable titers. Providinganti-ZP titers persist for 1 year, it is expected that females in theexperiment group will not conceive and therefore should not return toSable Island in 1994.

It should also be noted that fewer seals in the experimental groupreturned to Sable Island during the 1993 season (51 of 85 expected) thanthe control group (76 of 85 expected). These results demonstrate thatthe liposome vaccine was effective as an immunocontraceptive even in the1992 breeding season, immediately following administration of thevaccine. Anti-ZP antibodies inhibit implantation of embryos in theuterus. As seals have delayed implantation of about 3 months, antibodyproduction had sufficient time to build up to concentrations whichinhibited implantation (Henderson et al, 1988). This explanation issupported by recapture of three seals (#44, #65 and #66) which had beenradio tracked. Their sera had anti-ZP titers of 12, 34 and 56%,respectively, 6 months after being immunized. The seals that return toSable Island in 1994 will be further analyzed.

TABLE 4 Anti-ZP titers (as a % of a standard serum) in grey seals oneyear after injection with liposomes/FCA (controls) or ZP inliposomes/FCA (experimental) Controls Experimental Seal # Titer (%) Seal# Titer (%)  1 0  2 12  5 0  5 11  7 0  6 17  8 0  7 38 16 0  8 71 19 010  6 27 0 11  4 76 0 15  0 77 0 18 19 86 0 19 37 87 0 20  5 88 0 22 4194 0 27 21 96 0 29  3 103  0 31  2 112  0 33 67 117  0 34 20 35 27 39  841 32 42  4 47 16 48 58 52  5 53 42 55 12 57 25 61 10 62  3 63  4 65 5167  2 68  4 70 33 71 26 73  2 74 91 76 75 77  2 78 26 79 76 81 77 84 2988 26 91  5 93 16 94 26 96 49 101  10 104   6 *A value of 100% indicatesthe serum being tested has the same titer as a pooled standard serumfrom rabbits rendered infertile by immunization with SIZP.

EXPERIMENT 5

After the filing of the priority application (U.S. Ser. No. 07/892,807),the inventors continued their research to develop and improve thepreviously mentioned liposomal vaccine formulations incorporating zonapellucida (ZP) antigens. One new embodiment involved the incorporationof a freeze-dried liposome containing ZP and adjuvant into aBALLISTIVET* biobullet. A biobullet is a gelatin capsule adapted toincorporate antigens for the purpose of vaccinating large animals. Thebiobullet is shot into the animal with a specially designed gun thusavoiding the need to restrain and inject the animals.

Procedure

1. Preparation of Biobullet

The biobullets were obtained from BALLISTIVET (Minneapolis, Minn.) andpacked with the appropriate vaccine or control formulation as describedbelow.

2. Immunization of Rabbits

Rabbits (two rabbits for each treatment) were injected i.m. (a+b below)or a biobullet was surgically implanted (c below) as follows: (a) ZP (20μg) was encapsulated in liposomes prepared according to U.S. Pat. No.4,485,054 and the liposomes were suspended in a mixture of saline (0.25ml) and FCA (0.25 ml); (b) ZP (20 μg) encapsulated in liposomes preparedas above and suspended in a mixture of saline (0.25 ml) and FIA (0.25ml) and (c) ZP (20 μg) encapsulated in liposomes prepared as above thenfreeze-dried and the freeze-dried powder suspended in FCA (0.25 ml).This suspension was packed into a biobullet (BallistiVet) and implantedi.m. by surgery.

Results:

The results of this study are shown in Table 5. Administration of ZP inliposomes with FCA produced high anti-porcine ZP antibody titers withinone-two months. Replacing FCA with FIA, only marginally reduced anti-ZPantibody production. Use of freeze-dried liposomes with FCA in abiobullet resulted in an increased and prolonged production of anti-ZPantibodies.

TABLE 5 Effect of adjuvants on the production of anti-ZP antibodies byrabbits immunized with ZP in liposomes or freeze-dried liposomes inbiobullets. Titer (% of standard serum) Time (months) Delivery/adjuvant1 2 3 4 5 Liposome/FCA 120 131 103 24 16  38  38  24 17 19 Liposome/FIA 12  26  38  6 —  91  97  73 18 19 Biobullet/FCA 159 149 215 27 66  50196 344 128  98 FCA = Freund's complete adjuvant. FIA = Freund'sincomplete adjuvant.

EXPERIMENT 6

In this experiment, the three seals from Experiment 2 in groups (a) and(b) that were tested up until month 22 for anti-ZP antibodies were givena booster of SIZP (90 μg) in freeze-dried liposomes with Freund'sIncomplete Adjuvant (FIA) delivered with a BALLISTIVET biobullet intothe hindquarter of each seal. Serum samples were taken after 4 weeks(month 23), 6 weeks (month 23.5), 8 weeks (month 24) and 12 weeks (month25) after administration of the booster and anti-porcine ZP titersmeasured.

Results:

The results are shown in Table 6. A booster administered using abiobullet increased the anti-ZP antibodies to high levels which peaked6-8 weeks later. It is expected that at least one more serum sample willbe taken from these animals before they are released.

TABLE 6 Anti-ZP Titer (% of a standard serum) Time (months) Antigen 2323.5 24 25 ZP (15 μg) — — FCA/lipos 116 159 119 79 ZP (90 μg)  54  59 68 33 FCA/lipos  15  21  41 13

EXPERIMENT 7

The zone pellucida (ZP) antigen used in the previously mentioned sealstudies was porcine derived. Therefore in order to demonstrate thatantibodies raised in seals to porcine ZP do cross-react with sealoocytes, the following experiment was conducted.

Procedure:

Antibodies from unimmunized harp seals and harp seals immunized withporcine ZP were purified by affinity chromatography using a Protein Acolumn (Pierce Chemical Co., U.S.A.). The two antibody preparations werelabelled (¹⁴C) by reductive methylation (Jentoft, N. and D.G. Dearborn,1979, Labelling of proteins by reductive methylation using sodiumcyanoborohydride, J. Biol. Chem. 254: 4359-4365). Labelled antibodieswere incubated with either seal or porcine oocytes overnight at 2° C.Unbound antibody was removed by centrifugation with washing (2×'s) withsaline. Labelled antibody bound to oocytes was determined by liquidscintillation counting (LKB racbeta instrument). Oocytes were countedusing a microscope and a haemocytometer-like counting chamber.

Results:

The results are shown in Table 7. Seal and porcine oocytes incubatedwith labelled antibodies from control seals bound only small quantitiesof antibody (25-52 DPM).

Seal oocytes (93) bound ¹⁴C-labelled anti-porcine ZP antibodies, 119 DPM(35.8 ng protein/oocyte) whereas porcine oocytes (96) bound moreantibody (250 DPM; 72.9 ng protein/oocyte). This result suggests thatabout one-half the antibodies produced against porcine ZP in sealsrecognized and bound to seal ZP. Increasing the number of seal oocytesfrom 93 to 178 resulted in an increase in the quantity of antibody boundto oocytes (from 119 DPM to 169 DPM) demonstrating all sites on the sealoocytes capable of binding antibody were occupied. Increasing the numberof porcine oocytes from 96 to 192, 432 and 864 oocytes resulted in nosignificant increase in the quantity of ¹⁴C-labelled antibody bound(250, 228, 236 and 277 DPM). This result demonstrated that 96 porcineoocytes bound all anti-porcine-ZP antibody present in the serum from theimmunized seal.

These results verify that anti-porcine ZP antibodies bind to sealoocytes and provide evidence that immunocontraception may be explainedby the binding of antibodies to the oocyte surface.

TABLE 7 Binding of ¹⁴C-labelled anti-ZP antibodies to pig and sealoocytes Ab from control seal Ab from immunized seal Ab protein Abprotein oocytes Ab bound bound Ab bound bound source number (DPM)(ng/oocyte) (DPM) (ng/oocyte) seal 178 38 5.0 189 26.6 seal  93 25 6.3119 35.8 porcine 864 52 1.4 277 9.0 porcine 432 47 2.6 236 15.3 porcine192 45 5.5 228 33.3 porcine  96 55 13.6 250 72.9 The Ab from the controlseal added to the oocytes contained 2124 DPM (specific activity, 42,309DPM/mg protein) whereas the Ab from the immunized seal added to theoocytes contained 1672 DPM (specific activity, 35,685 DPM/mg protein).

SUMMARY

The above described experiments demonstrate that incorporating a zonapellucida antigen with a suitable adjuvant into a liposome deliverysystem results in an effective vaccine system for immunocontraception.The studies have shown that a single injection of the vaccine induceslevels of anti-zona pellucida antibodies up to at least 22 months. Asingle injection vaccine is clearly advantageous for anyimmunocontraceptive protocol.

Initial studies have shown that incorporating a freeze-dried liposomecontaining the ZP antigen and adjuvant into a biobullet also induceshigh levels of anti-ZP antibodies in the seals. Such a delivery systemis advantageous since it does not require that the animals be restrainedto be injected with the vaccine.

While the above described experiments relate to certain embodiments ofthe present invention, it is to be appreciated that variousmodifications can be made to the vaccine composition without departingfrom the scope and spirit of the invention. For example, while the abovedescribed examples relate to porcine ZP3, any suitable and effectivezona pellucida antigen, of porcine or other origin, may be employed. Infact, studies have shown that the genes that code for zona pellucida areconserved among mammals. The zona pellucida antigen may also be purifiedfrom oocytes or alternatively, a recombinant ZP antigen may be used.Modifications can also be made to the liposome delivery system. Inparticular, any liposomal system (such as freeze dried, liquid orsemi-solid forms) which allows for the slow, controlled release of theantigen is considered within the scope of the present invention.Finally, the adjuvant used can also be modified, as long as it iseffective in enhancing the immune response to the vaccine and issuitable for the intended application.

We claim:
 1. A method of preventing fertilization in a mammal for atleast twelve months which method consists of a single parenteraladministration of an effective amount of a vaccine compositioncomprising a zona pellucida derived antigen and a Freund's adjuvant in aliposome formulation.
 2. A method according to claim 1 wherein said zonapellucida derived antigen is ZP3.
 3. A method according to claim 1wherein said zona pellucida derived antigen is solubilized intact zonapellucida.
 4. A method according to claim 1 wherein said zona pellucidaderived antigen is porcine derived.
 5. A method according to claim 1wherein said liposome formulation is freeze-dried.
 6. A method accordingto claim 1 wherin said mammal is a seal.
 7. A method of preventingfertilization in a seal for at least 12 months which method consists ofa single parenteral administration of an effective amount of a vaccinecomposition comprising a zona pellucida derived antigen and an adjuvanteffective in enhancing the immune response to the vaccine composition,in a liposome formulation.
 8. The method according to claim 7 whereinsaid zona pellucida derived antigen is ZP
 3. 9. The method according toclaim 7 wherein said zona pellucida derived antigen is solubilizedintact zona pellucida.
 10. The method according to claim 7 wherein saidzona pellucida derived antigen is porcine derived.
 11. The methodaccording to claim 7 wherein said liposome formulation is freeze-dried.12. The method according to claim 7 wherein said adjuvant is Freund'sadjuvant.
 13. A method for preventing fertilization in a mammal for upto at least 22 months which method consists of a single parenteraladministration of an effective amount of a vaccine compositioncomprising a zona pellucida derived antigen and an adjuvant effective inenhancing the immune response to the vaccine composition, in a liposomeformulation.
 14. The method according to claim 13 wherein fertilizationis prevented for up to 22 months.
 15. The method according to claim 14wherein said zona pellucida derived antigen is ZP
 3. 16. The methodaccording to claim 14 wherein said zona pellucida derived antigen issolubilized intact zona pellucida.
 17. The method according to claim 14wherein said zona pellucida derived antigen is porcine derived.
 18. Themethod according to claim 14 wherein said liposome formulation isfreeze-dried.
 19. The method according to claim 14 wherein said mammalis a seal.
 20. The method according to claim 14 wherein said adjuvant isFreund's adjuvant.
 21. The method according to claim 13 wherein saidzona pellucida derived antigen is ZP
 3. 22. The method according toclaim 13 wherein said zona pellucida derived antigen is solubilizedintact zona pellucida.
 23. The method according to claim 13 wherein saidzona pellucida derived antigen is porcine derived.
 24. The methodaccording to claim 13 wherein said liposome formulation is freeze-dried.25. The method according to claim 13 wherein said mammal is a seal. 26.The method according to claim 13 wherein said adjuvant is Freund'sadjuvant.